Study On C9orf72 Regulation Of Stress Granule Dynamics

Background:G4C2 repeat expansions in chromosome 9 open reading frame 72（C9orf72）are the most common genetic cause of amyotrophic lateral sclerosis（ALS）and frontotemporal dementia（FTD）.The mutation appeared to produce both haploinsufficiency and gain-of-function effects in aggregating expanded RNAs and dipeptide repeat proteins（DPR）.The exact mechanism of the pathology of ALS/FTD is unknown.Recent studies have shown that abnormal stress granules（SGs）are related to neurodegenerative diseases,and expression of DPR or C9orf72 in cells induced spontaneous stress granule assembly.However,it is still unknown how DPR collaborative knock out C9orf72affects the dynamics of SGs and the consequences of abnormal regulation of SGs.Therefore,in this study,we constructed the models of knock out C9orf72,over-expression DPR,and DPR synergistic knock out C9orf72in cells,respectively,to investigate the relationship between the C9orf72and SGs by analyzing the dynamics of SGs and the level of SG-related protein expression.Objective:In this study,we aimed to explore whether the cell models of knock out C9orf72,over-expression DPR,and DPR synergistic knock out C9orf72 would cause any abnormal SGs dynamics and the underlying molecular or biological mechanisms.Met hods:（1）Lentiviral vector was synthesized by using the shRNA interference technique,and the cell model of shRNA-C9orf72 was constructed by transfecting HEK293T cells with the virus;（2）The co-localization of endogenous and exogenous C9orf72 and SGs were detected by immunofluorescence;（3）The influence of knock out C9orf72on SGs assembly and disassembly was investigated by immunofluorescence in different cells,and the SGs components and SGs related protein expression levels were detected by western blotting;（4）The vectors of GA₁₇₅-GFP,PR₁₇₅-GFP,and GR₁₄₉-GFP were constructed by designing and synthesizing different GGGGCC repeats,and the expression of DPR was detected by immunofluorescence;（5）HEK293T cells were transfected with GA₁₇₅-GFP,PR₁₇₅-GFP and GR₁₄₉-GFP vector respectively,and the co-localization of DPR and SGs was detected by immunofluorescence,the influence of over-expression DPR on SGs assembly and disassembly was investigated by immunofluorescence in HEK293T cells,which was transfected with DPR vector,after that,the SGs components and SGs related protein expression levels were detected by western blotting;（6）We investigated the influence of GA on nucleocytoplasmic transport by immunofluorescence;（7）HEK293T cells were co-transfected with shRNA-C9orf72 and DPR vectors,and the effect of DPR synergistic knock out C9orf72 on SGs dynamics was investigated by immunofluorescence,and the changes of DPR synergistic knock out C9orf72 in SGs components and SGs related protein expression levels were detected by western blotting.Results:（1）The cell lines of shRNA-C9orf72 was successfully obtained;（2）C9orf72 co-located with SG components protein G3BP1after sodium arsenite or heat shock stress treatment in HEK293T and He La cells;（3）Decreased expression of C9orf72 inhibited the assembly of SGs and accelerated the disassembly of SGs;（4）Decreased expression of C9orf72 up-regulated the level of TIA1,UBQLN2 and PSMD11proteins in shRNA-C9orf72 cell lines;（5）Decreased expression of C9orf72 up-regulated the phosphorylation of 4EBP1 and down-regulated the phosphorylation of e IF2αin shRNA-C9orf72 cell lines;（6）GR and PR co-located with SG components protein G3BP1 after sodium arsenite in HEK293T cells;（7）GR、PR and GA induce spontaneous assembly of poorly dynamic stress granules and impair translation;（8）The phosphorylation of e IF2αwas up-regulated when overexpression of DPR in HEK293T cells;（9）GA was found to to cause nucleocytoplasmic transport defects by destroying the complete structure of NPC and the nuclear input of Ran/Nup62;（10）DPR synergistic knock out C9orf72 in hibited the assembly of SGs and unchanged the disassembly of SGs.DPR synergistic knock out C9orf72 down-regulated the phosphorylation of e IF2αand up-regulated the phosphorylation of 4EBP1 in cells.Conclusion:In summary,decreased expression of C9orf72 inhibited the assembly of SGs and accelerated the disassembly of SGs,and decreased expression of C9orf72 affects the assembly of stress granules by down-regulating the phosphorylation of e IF2αand up-regulating the phosphorylation of 4EBP1.For the first time,we found that decreased expression of C9orf72 up-regulated the level of UBQLN2,TIA1.At the same time,we also found that expression of DPR in cells induced spontaneous stress granule assembly that required e IF2αphosphorylation.Intriguingly,we found that DPR synergistic knock out C9orf72 inhibited the assembly of SGs.This abnormal phenomenon may be caused by decreased phosphorylation level of e IF2αand increased phosphorylation level of 4EBP1 in cells.